Excessive scarring of corneal wounds caused by trauma, infectious diseases or refractive surgery leads to impaired vision. Attempts to reduce scarring with anti-inflammatory steroids or anti-metabolites (5FU, mitomycin-C) can cause side effects and chronically reduce corneal keratocyte density. Our long term goals are to understand the molecular regulation of corneal scarring and then design gene-targeted approaches to reduce scarring. In our previous grant, we focused on biochemically defining the roles of two key fibrogenic growth factors, TGFp and CTGF, in corneal scarring. We showed that TGFp up-regulated expression of CTGF and that CTGF mediated TGFp-stimulated collagen synthesis and matrix contraction in cultures of human corneal fibroblasts (HCF). We identified the CTGF receptor (M6P/IGF-II-R) and found that CTGF mRNA, protein and receptor levels all dramatically increased during healing of excimer laser injuries of rat corneas. We showed that the MEK1/2, ERK1/2, STATS cascade is a major signaling pathway for CTGF in HCF. Affymetrix microarray analyses of excimer ablated rat corneas showed major changes in patterns of gene expression including corneal crystallins, TGFp and CTGF. We developed ribozymes and antisense oligonucleotides that selectively reduced TGFp and CTGF mRNA and protein levels in HCF cultures and established conditions for AAV vector transduction in rabbit corneas. In this new grant, we will extend these results to further understand the mechanism and physiological roles of TGFp and CTGF in corneal scarring, including the recently discovered proteolytic cleavage of CTGF into a N-terminal fragment that stimulates cell proliferation and a C-terminal fragment that stimulates collagen synthesis. We will (1) identify the cellular protease that cleaves CTGF into N/C terminal fragments; (2) characterize the ratio of N/C terminal proteins during healing of excimer ablation wounds; (3) identify receptors for the N/C-terminal fragments and establish their signal transduction pathways in HCF; (4) fully characterize gene expression patterns for N/C- terminal proteins in HCF and CTGF knockout fibroblasts (Affymetrix microarray); (5) optimize knock down of TGFp and CTGF mRNAs and proteins using a ribozyme and shRNA combination; (6) evaluate combined ribozyme and shRNA expression from a self complementary AAV (scAAV) vector that allows rapid turn on with high expression of transgenes in a rabbit model of corneal excimer laser wound healing. These integrated experiments will expand the understanding of molecular regulation of corneal scarring by the TGFp and CTGF systems and evaluate translation of gene-targeted therapy to control corneal scarring. ? ? ?
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